Effects of Na+/Ca2+-exchanger Overexpression on Excitation–contraction Coupling in Adult Rabbit Ventricular Myocytes

doi:10.1006/jmcc.2001.1521

Journal of Molecular and Cellular Cardiology

Volume 34, Issue 4, April 2002, Pages 389-400

https://doi.org/10.1006/jmcc.2001.1521 Get rights and content

Abstract

The Na⁺/Ca²⁺-exchanger (NCX) is the main mechanism by which Ca²⁺ is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation–contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation–contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca²⁺ could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca²⁺ entry via the L-type Ca²⁺ channel. Systolic and diastolic Ca²⁺ levels were significantly reduced, with peak systolic Ca²⁺ in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca²⁺. Both cell relengthening and the decay of the Ca²⁺ transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca²⁺ stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca²⁺ stores and low diastolic Ca²⁺ levels rather than reduced Ca²⁺ entry.

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New experimental evidence for mechanism of arrhythmogenic membrane potential alternans based on balance of electrogenic I<inf>NCX</inf>/I <inf>Ca</inf> currents
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INCX predominance was achieved by in vivo NCX gene transfer by using a modified cross-clamping method.9 Western blot from in vitro NCX overexpression showed that NCX protein expression was indeed increased by (3.8 ± 2.9)-fold compared with control (n = 3), and previously Ranu et al10 demonstrated that the overexpression of NCX increases INCX, with no change in ICa. ICa predominance was achieved by INCX inhibition or ICa enhancement.
Computer simulations have predicted that the balance of various electrogenic sarcolemmal ion currents may control the amplitude and phase of beat-to-beat alternans of membrane potential (V_m). However, experimental evidence for the mechanism by which alternans of calcium transients produces alternation of V_m (V_m-ALT) is lacking.
To provide experimental evidence that Ca-to-V_m coupling during alternans is determined by the balanced influence of 2 Ca-sensitive electrogenic sarcolemmal ionic currents: I_NCX and I_Ca.
V_m-ALT and Ca-ALT were measured simultaneously from isolated guinea pig myocytes (n = 41) by using perforated patch and Indo-1_AM fluorescence, respectively. There were 3 study groups: (1) control, (2) I_NCX predominance created by adenoviral-induced NCX overexpression, and (3) I_Ca predominance created by I_NCX inhibition (SEA-0400) or enhanced I_Ca (As₂O₃). During alternans, 14 of 14 control myocytes demonstrated positive Ca-to-V_m coupling, consistent with I_NCX, but not I_Ca, as the major electrogenic current in modulating action potential duration. Positive Ca-to-V_m coupling was maintained during I_NCX predominance in 8 of 8 experiments with concurrent increase in Ca-to-V_m gain (P <.05), reaffirming the role of increased forward-mode electrogenic I_NCX. Conversely, I_Ca predominance produced negative Ca-to-V_m coupling in 14 of 19 myocytes (P < .05) and decreased Ca-to-V_m gain compared with control (P <.05). Furthermore, computer simulation demonstrated that Ca-to-V_m coupling changes from negative to positive because of a shift from I_Ca to I_NCX predominance with increasing pacing rate.
These data provide the first direct experimental evidence that coupling in phase and magnitude of Ca-ALT to V_m-ALT is strongly determined by the relative balance of the prominence of I_NCX vs I_Ca currents.
Systematic characterization of the ionic basis of rabbit cellular electrophysiology using two ventricular models
2011, Progress in Biophysics and Molecular Biology
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Many experimental results support the primary role of GCaL, GNaK and GNaCa on systolic [Ca2+]i shown in our simulations. The important role played by GNaK in modulating systolic [Ca2+]i levels in both rabbit models is supported by the marked increase in cell force of contraction and calcium levels caused by INaK inhibitors, such as ouabain (Giles and Shimoni, 1989; Kodama et al., 1981; McCans et al., 1974; Schillinger et al., 2003) and strophantidine (Loughrey et al., 2003; Ranu et al., 2002; Szigligeti et al., 1996). Furthermore, experimental results showing reduction of cell contraction (related to systolic [Ca2+]i) caused by the ICaL blocker verapamil (Mahajan et al., 2008a), are consistent with the importance of GCaL in determining systolic [Ca2+]i levels.
Several mathematical models of rabbit ventricular action potential (AP) have been proposed to investigate mechanisms of arrhythmias and excitation-contraction coupling. Our study aims at systematically characterizing how ionic current properties modulate the main cellular biomarkers of arrhythmic risk using two widely-used rabbit ventricular models, and comparing simulation results using the two models with experimental data available for rabbit. A sensitivity analysis of AP properties, Ca²⁺ and Na⁺ dynamics, and their rate dependence to variations (±15% and ±30%) in the main transmembrane current conductances and kinetics was performed using the Shannon et al. (2004) and the Mahajan et al., 2008a, Mahajan et al., 2008b AP rabbit models. The effects of severe transmembrane current blocks (up to 100%) on steady-state AP and calcium transients, and AP duration (APD) restitution curves were also simulated using both models. Our simulations show that, in both virtual rabbit cardiomyocytes, APD is significantly modified by most repolarization currents, AP triangulation is regulated mostly by the inward rectifier K⁺ current (I_K1) whereas APD rate adaptation as well as [Na⁺]_i rate dependence is influenced by the Na⁺/K⁺ pump current (I_NaK). In addition, steady-state [Ca²⁺]_i levels, APD restitution properties and [Ca²⁺]_i rate dependence are strongly dependent on I_NaK, the L-Type Ca²⁺ current (I_CaL) and the Na⁺/Ca²⁺ exchanger current (I_NaCa), although the relative role of these currents is markedly model dependent. Furthermore, our results show that simulations using both models agree with many experimentally-reported electrophysiological characteristics. However, our study shows that the Shannon et al. model mimics rabbit electrophysiology more accurately at normal pacing rates, whereas Mahajan et al. model behaves more appropriately at faster rates. Our results reinforce the usefulness of sensitivity analysis for further understanding of cellular electrophysiology and validation of cardiac AP models.
Targeted GLUT-4 deficiency in the heart induces cardiomyocyte hypertrophy and impaired contractility linked with Ca<sup>2+</sup> and proton flux dysregulation
2010, Journal of Molecular and Cellular Cardiology
There is clinical evidence to suggest that impaired myocardial glucose uptake contributes to the pathogenesis of hypertrophic, insulin-resistant cardiomyopathy. The goal of this study was to determine whether cardiac deficiency of the insulin-sensitive glucose transporter, GLUT4, has deleterious effect on cardiomyocyte excitation–contraction coupling. Cre-Lox mouse models of cardiac GLUT4 knockdown (KD, 85% reduction) and knockout (KO, > 95% reduction), which exhibit similar systemic hyperinsulinemic and hyperglycemic states, were investigated. The Ca²⁺ current (I_Ca) and Na⁺–Ca²⁺ exchanger (NCX) fluxes, Na⁺–H⁺ exchanger (NHE) activity, and contractile performance of GLUT4-deficient myocytes was examined using whole-cell patch-clamp, epifluorescence, and imaging techniques. GLUT4-KO exhibited significant cardiac enlargement characterized by cardiomyocyte hypertrophy (40% increase in cell area) and fibrosis. GLUT4-KO myocyte contractility was significantly diminished, with reduced mean maximum shortening (5.0 ± 0.4% vs. 6.2 ± 0.6%, 5 Hz). Maximal rates of shortening and relaxation were also reduced (20–25%), and latency was delayed. In GLUT4-KO myocytes, the I_Ca density was decreased (− 2.80 ± 0.29 vs. − 5.30 ± 0.70 pA/pF), and mean I_NCX was significantly increased in both outward (by 60%) and inward (by 100%) directions. GLUT4-KO expression levels of SERCA2 and RyR2 were reduced by approximately 50%. NHE-mediated H⁺ flux in response to NH₄Cl acid loading was markedly elevated GLUT4-KO myocytes, associated with doubled expression of NHE1. These findings demonstrate that, independent of systemic endocrinological disturbance, cardiac GLUT4 deficiency per se provides a lesion sufficient to induce profound alterations in cardiomyocyte Ca²⁺ and pH homeostasis. Our investigation identifies the cardiac GLUT4 as a potential primary molecular therapeutic target in ameliorating the functional deficits associated with insulin-resistant cardiomyopathy.
β-Adrenergic receptor stimulated Ncx1 upregulation is mediated via a CaMKII/AP-1 signaling pathway in adult cardiomyocytes
2010, Journal of Molecular and Cellular Cardiology
Citation Excerpt :
Transgenic overexpression of the cytosolic splice variant, CaMKIIδc, induces severe heart failure associated with SR Ca2+ leak and reduced SR Ca2+ content [57]. Correspondingly, the upregulation of Ncx1 contributes directly to limiting SR loading, contractile dysfunction and greater potential for delayed after depolarizations, which lead to ventricular tachycardia [9,18–20]. Furthermore, numerous studies of human and animal models of heart failure demonstrate that diastolic performance in the failing heart correlates inversely with protein levels of NCX1 [17,20,59].
The Na⁺–Ca²⁺ exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. β-Adrenergic receptor (β-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca²⁺/calmodulin-dependent protein kinase II (CaMKIIδ_c) null mouse, we demonstrate that β-AR-stimulated Ncx1 upregulation is dependent on CaMKII. β-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that β-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically β-AR-stimulated heart.
Chronic administration of KB-R7943 induces up-regulation of cardiac NCX1
2009, Journal of Biological Chemistry
The NCX1 (sodium-calcium exchanger) is up-regulated in human heart failure and in many animal models of heart failure. The potential benefits and risks of therapeutically blocking NCX1 in heart failure and during ischemia-reperfusion are being actively investigated. In this study, we demonstrate that prolonged administration of the NCX1 inhibitor KB-R7943 resulted in the up-regulation of Ncx1 gene expression in both isolated adult cardiomyocytes and intact mouse hearts. Ncx1 up-regulation is mediated by the activation of p38. Importantly, p38 is not activated by KB-R7943 treatment in heart tubes from Ncx1^−/− mice at 9.5 days postcoitum but is activated in heart tubes from Ncx1^+/+ mice. p38 activation does not appear to be in response to changes in cytosolic calcium concentration, [Ca²⁺]_i. Interestingly, chronic KB-R7943 treatment in mice leads to the formation of an NCX1-p38 complex. Our study demonstrates for the first time that the electrogenic sarcolemma membrane cardiac NCX1 can act as a regulator of “activity-dependent signal transduction” leading to changes in gene expression.

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^f1: Please address all correspondence to: Dr Sian E. Harding, Cardiac Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse StLondon SW3 6LY, UK. Tel: 0044 207 351 8146. Fax: 0044 207 823 3392. E-mail: [email protected]

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